<p>Midgut carcinoid tumours are rare with an incidence of 0.5-2.1/100 000. The primary tumour is usually small and grows slowly but has almost always set metastases at diagnosis. When radically operated, most patients will eventually recur in their disease. </p><p>We evaluated different methods in detecting recurrent disease in 61 malignant midgut carcinoid tumours that had been radically operated. Thirty-eight patients have been diagnosed with a recurrence. In 32/38 of these patients P-Chromogranin A was the first method to indicate a recurrence. We therefore recommend using P-CgA in the work up in these patients.</p><p>We investigated characteristics, survival and independent factors that could be of bad prognostic value. We found that in our 284 malignant midgut carcinoid tumours, 208/284 (73%) had distant metastases and 30/284 (11%) had carcinoid heart disease. Median survival was 115.5 months and five-year survival was 77%. In a multivariate analysis liver metastases and carcinoid heart disease were poor prognostic factors.</p><p>We performed a phase II study with octreotide pamoate investigating the clinical effect in 12 malignant midgut carcinoid tumours in a progressive phase. We found that 9/12 (75%) were stabilised for a median duration of 12 months. We think that this is a good effect considering the advanced stage.</p><p>We investigated the frequency of four different tyrosine kinase receptors, platelet derived growth factor receptor (PDGR) α and β, epidermal growth factor receptor (EGFR) and c-kit, in 36 malignant midgut carcinoid tumours with immunohistochemistry. We found that 13/34 (38%) tumour samples expressed PDGFRα, 29/33 (88%) PDGFRβ, 24/33 (73%) EGFR, whereas none expressed c-kit. This implicates that midgut carcinoid tumours might be susceptible to treatment with tyrosine kinase receptor inhibitors. </p>

Midgut carcinoid tumours are rare with an incidence of 0.5-2.1/100 000. The primary tumour is usually small and grows slowly but has almost always set metastases at diagnosis. When radically operated, most patients will eventually recur in their disease. We evaluated different methods in detecting recurrent disease in 61 malignant midgut carcinoid tumours that had been radically operated. Thirty-eight patients have been diagnosed with a recurrence. In 32/38 of these patients P-Chromogranin A was the first method to indicate a recurrence. We therefore recommend using P-CgA in the work up in these patients. We investigated characteristics, survival and independent factors that could be of bad prognostic value. We found that in our 284 malignant midgut carcinoid tumours, 208/284 (73%) had distant metastases and 30/284 (11%) had carcinoid heart disease. Median survival was 115.5 months and five-year survival was 77%. In a multivariate analysis liver metastases and carcinoid heart disease were poor prognostic factors. We performed a phase II study with octreotide pamoate investigating the clinical effect in 12 malignant midgut carcinoid tumours in a progressive phase. We found that 9/12 (75%) were stabilised for a median duration of 12 months. We think that this is a good effect considering the advanced stage. We investigated the frequency of four different tyrosine kinase receptors, platelet derived growth factor receptor (PDGR) α and β, epidermal growth factor receptor (EGFR) and c-kit, in 36 malignant midgut carcinoid tumours with immunohistochemistry. We found that 13/34 (38%) tumour samples expressed PDGFRα, 29/33 (88%) PDGFRβ, 24/33 (73%) EGFR, whereas none expressed c-kit. This implicates that midgut carcinoid tumours might be susceptible to treatment with tyrosine kinase receptor inhibitors.

Carvacrol is a phenol monoterpene found in aromatic plants specially in Lamiaceae family, which has been evaluated in an experimental model of breast cancer. However, any proposed mechanism based on its antitumor effect has not been reported. In our previous study, carvacrol showed a protective effect on 7,12-dimethylbenz[α]anthracene- (DMBA-) induced breast cancer in female rats. The main objective in this research was to evaluate by using in silico study the carvacrol on HER2, PI3Kα, mTOR, hERα, PR, and EGFR receptors involved in breast cancer progression by docking analysis, molecular dynamic, and drug-likeness evaluation. A multilevel computational study to evaluate the antitumor potential of carvacrol focusing on the main targets involved in the breast cancer was carried out. The in silico study starts with protein-ligand docking of carvacrol followed by ligand pathway calculations, molecular dynamic simulations, and molecular mechanics energies combined with the Poisson–Boltzmann (MM/PBSA) calculation of the free energy of binding for carvacrol. As result, the in silico study led to the identification of carvacrol with strong binding affinity on mTOR receptor. Additionally, in silico drug-likeness index for carvacrol showed a good predicted therapeutic profile of druggability. Our findings suggest that mTOR signaling pathway could be responsible for its preventive effect in the breast cancer. / Revisión por pares

Oocyte maturation (OM) in teleosts is under precise hormonal control by estrogens and progestins. We show here that estrogens activate an epidermal growth factor receptor (EGFR) signaling pathway through the G protein-coupled estrogen receptor (GPER) to maintain meiotic arrest of full-grown zebrafish (Danio rerio) oocytes in an in vitro germinal vesicle breakdown (GVBD) bioassay. A GPER- specific agonist decreased OM and a GPER-specific antagonist increased spontaneous OM, whereas specific nuclear estrogen receptor (ERα and ERβ) agonists did not affect OM, which suggests the inhibitory action of estrogens on OM are solely mediated through GPER. Furthermore, a peptide-bound estrogen, which cannot enter the oocyte, decreased GVBD, showing that these estrogen actions are mediated through a membrane receptor. Treatment of oocytes with actinomycin D, a transcription inhibitor, did not block the inhibitory effects of estrogens on OM, indicating that estrogens act via a nongenomic mechanism to maintain oocyte meiotic arrest. EGFR mRNA was detected in denuded zebrafish oocytes by reverse transcription polymerase chain reaction (RT-PCR). Therefore, the potential role of transactivation of EGFR in estrogen inhibition of OM was investigated. The matrix metalloproteinase inhibitor, ilomastat, which prevents the release of heparin-bound epidermal growth factor (HB-EGF), increased spontaneous OM. Moreover, specific EGFR1 (ErbB1) inhibitors and inhibitors of extracellular-related kinase 1 and 2 (ERK1/2) increased spontaneous OM. Previously, estrogens have been shown to increase 3’-5’-cyclic adenosine mono phosphate (cAMP) levels through GPER in zebrafish oocytes during meiotic arrest. Taken together these present results suggest that estrogens also act through GPER to maintain meiotic arrest through a second signaling pathway involving transactivation of EGFR and activation of ERK 1 and 2. / text

The introduction of tyrosine kinase inhibitors (TKI) targeting the epidermal growth factor receptor (EGFR) inhibitors to the clinic has resulted in an improvement in the treatment of non small cell lung cancer (NSCLC). However, many patients treated with EGFR TKIs do not respond to therapy. The burden of failed treatment is largely placed on the healthcare field, limiting the effectiveness of EGFR TKIs. Furthermore, responses are hindered by the emergence of resistance. Thus, two questions must be addressed to achieve maximum benefit of EGFR inhibitors: How can patients who will benefit from EGFR TKIs be selected a priori? How can patients who respond achieve maximal benefit? To answer these questions, two hypotheses were formed. First, the EGFR-dependent phenotype, which is displayed by the tumors cells of those patients who respond clinically to EGFR TKIs, can be captured by genomic profiling of NSCLC cell lines stratified by sensitivity to EGFR TKIs. This gene signature may be used to predict the outcome of EGFR TKI therapy in unknown samples. Secondly, the predictive signature of response to EGFR TKI could provide insights into the underlying biology of the phenotype of EGFR-dependency. This information could be exploited to identify inhibitors which could be combined with EGFR inhibitors to elicit a greater effect, thereby minimizing resistance. The work herein describes the testing of these hypotheses. Pharmacogenomics was utilized to define a signature of EGFR-dependency which effectively predicted response to EGFR TKI in vitro and in vivo. Furthermore, the signature was analyzed by bioinformatic approaches to identify the RAS/MAPK pathway as a candidate target in EGFR-dependent NSCLC. The RAS/MAPK pathway regulates expression and activation of EGF-like ligands. Furthermore, the RAS/MAPK pathway modulates EGFR stability in the EGFR-dependent phenotype. Further biochemical analyses demonstrated that the RAS/MAPK pathway mediates proliferation and survival of EGFR-dependent NSCLC cells. Finally, combinatorial treatment of EGFR-dependent NSCLC cell lines with small molecules targeting EGFR and the RAS/MAPK pathway yielded cytotoxic synergy. Thus, we have used pharmacogenomics methods to potentially improve NSCLC treatment by developing a method of predicting response and identifying an additional target to combine with EGFR TKIs to maximize responses.

Feline oral squamous cell carcinomas (FOSCCs) are locally aggressive tumours and a common cause of mortality and morbidity. Current treatment options are rarely successful and animals are frequently euthanised upon diagnosis due to their grave prognosis. Epidermal Growth Factor Receptor (EGFR) is a tyrosine kinase receptor which is frequently dysregulated in SCC of the head and neck (HNSCC) in man. Recent advances in human medicine have identified EGFR as a therapeutic target in HNSCC. In this study the role of EGFR in FOSCC was investigated. Sixty seven biopsy samples were immunohistochemically labelled for EGFR and Ki67, a proliferation marker. The tyrosine kinase region of feline EGFR was cloned and sequenced, and six small interfering RNAs (siRNAs) targeting the tyrosine kinase region were developed. The most effective siRNA as well as an EGFR specific tyrosine kinase inhibitor, gefitinib, was then used on a feline SCC cell line (SCCF1), and the effect of EGFR targeting alone, or in combination with irradiation, on the cell line was determined. The majority of the biopsy samples were labelled positively for EGFR and Ki67, and high proliferation corresponded with poor prognosis. The siRNA caused reduction in EGFR mRNA by Real-Time Polymerase Chain Reaction and protein levels as assessed by western blot analysis. Reduced cell proliferation and migration were also observed by proliferation assays and scratch assays respectively. Combining EGFR knockdown with irradiation caused an additive effect on the ability of the cell line to form colonies. These results support the role of EGFR as a potential therapeutic target in FOSCCs.

Cell polarity is an important regulator of cellular processes and is vital in helping to prevent metaplasia and tumorigenesis. There are three many polarity complexes that regulate and maintain epithelial cellular polarity. The Par and Crumbs complexes locate to the apical membrane of the cell, while the Scribble complex is located basolaterally. Of the Scribble complex components, the polarity protein Hugl1, also known as Mgl1 in mice, is especially important in helping to maintain apical basolateral and planar polarity, and is lost in multiple types of cancer. When Hugl1 expression is lost in epithelial cells, it results in a mesenchymal phenotype. We now show that the loss of Hugl1 fundamentally shifts the cellular phenotype and specifically alters EGFR trafficking and signaling. Loss of Hugl1 results in the nuclear translocation of Taz and Slug, increased migration, and the mislocalization of EGFR (Epidermal Growth Factor Receptor), driving cellular growth. Hugl1 regulates the expression of multiple cell identity markers and its loss results in stem cell characteristics, including the increased expression of CD44, and a decrease of CD49f and CD24 expression. The loss of Hugl1 also results in increased growth in soft-agar and prolonged survival when transplanted into NOD-SCID mice; its loss also results in EGF-dependent migration which aids in increasing mammosphere survival. Furthermore, isolated EGFR mislocalization via a point mutation (P667A) also drives these same phenotypes, including activation of Akt and Taz nuclear translocation, indicating the importance of Hugl1 in the regulation of EGFR localization and its signaling. In mice, the loss of total Mgl1 is lethal within days of birth due to hydrocephaly and results in the formation of rosette like structures in the brain that are reminiscent of neuroectodermal tumors. We designed a targeted Mgl1 knockout in the mammary epithelial cells using the Cre/Lox system to evaluate the effects of Mgl1 loss in murine mammary gland development and tumorigenesis. The loss of Mgl1 expression in mice inhibits ductal outgrowth, increases side branching and epithelial layers, and results in the mislocalization of EGFR. While overt mammary tumors did not develop, some individuals did develop hyperplastic nodules that could progress into cancer. The knockdown of Hugl1 in vitro and Mgl1 in vivo reveal how the loss of polarity and presence of Hugl1 results in cancer stem cell characteristics, increased migration, and abnormal signaling due to the mislocalization of EGFR. While these changes result in metaplasia and a potential pre-cancerous state, the loss of Hugl1 alone is not enough to drive the cancer progression, indicating that other mutations or factors are necessary for the development of breast cancer. Because of the key role polarity plays in the prevention of breast cancer development we investigated if the addition of Hugl1 back into breast cancer cells could revert the cancerous cells to a normal epithelial phenotype. Most of the breast cancer cells transfected with Hugl1 expression did not survive, indicating that the re-expression of polarity regulators forces cancer cells to die. The small percentage of cells that did survive re-expression of Hugl1 had retarded growth in soft agar and a decrease in EGFR expression. Together, these data indicate that Hugl1 expression and EGFR activity are closely related and that Hugl1 is required for the proper localization and signaling of EGFR. When Hugl1 is lost, EGFR is mislocalized and fails to be degraded properly, promoting pre-neoplastic changes.

A unique computational approach was undertaken to identify targets of miR-17-3p that impart an oncogenic potential to the cells of the prostate. Utilizing this approach, we identified insulin growth factor receptor 1 (IGF1R) as a potential target of miR-17-3p. IGF1R imparts an oncogenic approach to the cells by helping cells escape apoptosis, become hypertrophic and increase the production of extracellular proteases that allow cells to detach from neighbors. The regulation of insulin growth factor receptor 1 by human microRNA-17-3p was evaluated using a western blot analysis of prostate cancer cell lines. Protein levels were compared in a cell line that expressed a non-targeting control RNA and a cell line that expressed microRNA-17-3p. The cell line that expressed the non-targeting control had significantly higher levels of IGF1R protein than the cell line expressing more of the active microRNA. Based on this experiment, it appears that microRNA-17-3p might regulate the insulin growth factor receptor 1.

Single nucleotide polymorphisms present in the second intron of the fibroblast growth factor receptor 2 (FGFR2) gene have been linked with increased risk of breast cancer in several genome wide association studies. The potential effect of those SNPs appeared to be mediated through the differential binding of cis-regulatory elements, such as transcription factors, since all the SNPs in linkage disequilibrium were located in a regulatory DNA region. Preliminary studies have shown that a Runx2 binding site is functional only in the minor, disease associated allele of rs2981578, resulting in increased expression of FGFR2 in cancers from patients homozygous for that allele. Moreover, the increased risk conferred by the minor FGFR2 allele is associated most strongly in oestrogen receptor alpha positive (ERα) breast tumours, suggesting a potential interaction between ERα and FGFR signalling. Here, we have developed a human cell line model system to study the effect of those SNPs on cell behaviour. In an ERα positive breast cancer cell line, rs2981578 was edited using Zinc Finger Nucleases. Unexpectedly, the acquisition of the single risk allele in MCF7 cells failed to affect proliferation or cell cycle progression. Binding of Runx2 to the risk allele was not observed. However FOXA1 binding, an important ERα partner, appeared decreased at the rs2981578 locus in the risk allele cells. Additionally, differences in allele specific expression (ASE) of FGFR2 were not observed in a panel of 72 ERα positive breast cancer samples. Thus, the apparent increased risk of developing ERα positive breast cancer is not caused by rs2981578 alone. Rather, the observed increased risk of developing breast cancer might be the result of a coordinated effect of multiple SNPs forming a risk haplotype in the second intron of FGFR2.

Mechanistic insights on molecular and cellular mechanisms whereby KSHV induces Kaposi?s sarcoma (KS) are key for our understanding of KS tumors and for the development of new therapies. We have previously developed an animal model for KSHV induced KS using murine bone marrow cells transfected with a KSHVBac36. We found that although these cells lacked attributes of transformed cells in vitro, they were able to cause KS-like tumors in vivo. In vivo tumorigenesis correlated with upregulation of both KSHV lytic genes and host angiogenesis suggesting that that cues provided from the microenvironment played a major role in regulating viral and host gene expression related with KSHV-induced tumorigenesis. Our goal thus, was to identify these molecular cues regulating pathogenic gene expression in KSHV infected cells in vivo. An important difference between cells kept in vitro versus in vivo is the lack of environmental extracellular matrix (ECM) signals. Therefore the mECK36 cells were cultured in vitro in matrigel, a basement membrane preparation rich in ECM proteins and its individual components, to discern the effect of host signaling by the ECM on KSHV infected cells. Investigation of gene expression through Real Time RT-PCR identified several viral and host genes associated with tumorigenesis such as KSHV vGPCR and angiogenesis associated VEGF and EGF- receptors were upregulated in response to this environment. Further analysis of the molecular activity of the cell indicated the change in transcription was due to the activation of integrin signaling, as assessed by phosphorylation of the Focal Adhesion Kinase (FAK) protein. Our results show that integrin signaling occurring in vivo through interaction with ECM serves to enhance the pathogenic viral and host gene expression of KSHV infected cells and that EGFR upregulation can be correlated with these conditions. These results points to the integrin signaling pathway or the EGF-Receptor as promising targets for therapy and prevention of KS tumors.